Indole acetic acid acyl guanidines as β-secretase inhibitors

ABSTRACT

There is provided a series of substituted acyl guanidines of Formula (I) 
                         
or a stereoisomer; or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8  as defined herein, their pharmaceutical compositions and methods of use. These compounds inhibit the processing of amyloid precursor protein (APP) by β-secretase and, more specifically, inhibit the production of  A β-peptide. The present disclosure is directed to compounds useful in the treatment of neurological disorders related to β-amyloid production, such as Alzheimer&#39;s disease and other conditions affected by anti-amyloid activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a non-provisional application which claims the benefit of U.S. Provisional Application No. 60/713,316 filed Sep. 1, 2005.

FIELD OF THE DISCLOSURE

This patent application provides substituted indolyl acyl guanidines having drug and bio-affecting properties, their pharmaceutical compositions and method of use. In particular, the disclosure is concerned with a series of indolyl acyl guanidines which are inhibitors of β-amyloid peptide (β-AP) production, thereby acting to prevent the accumulation of amyloid protein deposits in the brain and, therefore, are useful in the treatment of neurological disorders related to β-amyloid production. More particularly, the present disclosure relates to the treatment of Alzheimer's Disease (AD) and similar diseases.

BACKGROUND

Alzheimer's Disease is a progressive, neurodegenerative disorder characterized by memory impairment and cognitive dysfunction. AD is characterized pathologically by the accumulation of senile (neuritic) plaques, neurofibrillary tangles, amyloid deposition in neural tissues and vessels, synaptic loss, and neuronal death. It is the most common form of dementia and it now represents the third leading cause of death after cardiovascular disorders and cancer. The cost of Alzheimer's Disease is enormous (in the U.S., greater than $100 billion annually) and includes the suffering of the patients, the suffering of families, and the lost productivity of patients and caregivers. As the longevity of society increases, the occurrence of AD will markedly increase. It is estimated that more than 10 million Americans will suffer from AD by the year 2020, if methods for prevention and treatment are not found. Currently, AD is estimated to afflict 10% of the population over age 65 and up to 50% of those over the age of 85. No treatment that effectively prevents AD or reverses the clinical symptoms and underlying pathophysiology is currently available (for review see Selkoe, D. J. Ann. Rev. Cell Biol., 1994, 10: 373-403).

Histopathological examination of brain tissue derived upon autopsy or from neurosurgical specimens in affected individuals reveals the occurrence of amyloid plaques and neurofibrillar tangles in the cerebral cortex of such patients. Similar alterations are observed in patients with Trisomy 21 (Down's syndrome). Biochemical and immunological studies reveal that the dominant proteinaceous component of the amyloid plaque is an approximately 4.2 kilodalton (kD) protein of about 39 to 43 amino acids. This protein is designated Aβ, β-amyloid peptide, and sometimes β/A4; referred to herein as Aβ. In addition to its deposition in amyloid plaques, Aβ is also found in the walls of meningeal and parenchymal arterioles, small arteries, capillaries, and sometimes, venules. Compelling evidence accumulated during the last decade reveals that Aβ is an internal polypeptide derived from a type 1 integral membrane protein, termed β-amyloid precursor protein (APP) (Selkoe, D. Physiol. Rev. 2001, 81, 741-766; Wolfe, M. J. Med. Chem. 2001, 44, 2039-2060). βAPP is normally produced by many cells both in vivo and in cultured cells, derived from various animals and humans. Several proteolytic fragments of APP are generated by proteinases referred to as secretases. A subset of these proteolytic fragments, designated β-amyloid peptide (Aβ), contains 39 to 43 amino acids and is generated by the combined action of β-secretase and γ-secretase. β-secretase is a membrane-bound, aspartyl protease that forms the N-terminus of the Aβ peptide. The C-terminus of the Aβ peptide is formed by γ-secretase, an apparently oligomeric complex that includes presenilin-1 and/or presenilin-2. Presenilin-1 and presenilin-2 are polytopic membrane-spanning proteins that may contain the catalytic components of γ-secretase (Seiffert, D.; Bradley, J. et al., J. Biol. Chem. 2000, 275, 34086-34091).

In addition to AD, excess production and/or reduced clearance of Aβ causes cerebral amyloid angiopathy (CAA) (reviewed in Thal, D., Gherbremedhin, E. et al., J. Neuropath. Exp. Neuro. 2002, 61, 282-293). In these patients, vascular amyloid deposits cause degeneration of vessel walls and aneurysms that may be responsible for 10-15% hemorrhagic strokes in elderly patients. As in AD, mutations in the gene encoding Aβ lead to an early onset form of CAA, referred to as cerebral hemorrhage with amyloidosis of the Dutch type, and mice expressing this mutant protein develop CAA that is similar to patients.

A logical approach to reducing Aβ levels is to interfere with the action of the secretases that are directly involved in the cleavage of APP to Aβ. The β-secretase enzyme (BACE) is responsible for cleaving APP and forms the amino-terminus of Aβ, initiating the amyloidogenic pathway. The BACE enzyme is a transmembrane aspartyl protease and was described in the literature by several independent groups [see Hussain, I. et al., (1999) Mol. Cell. Neurosci., 14: 419-427; Lin, X. et al., (2000) Proceedings of the National Academy of Sciences of the United States of America, 97: 1456-1460; Sinha, S., et al., (1999) Nature (London), 402: 537-540; Vassar, R., et al., (1999) Science (Washington, D.C.), 286: 735-741; Walsh, D. M. et al., (2002); Wolfe, M. S. (2001); Yan, R. et al., (1999) Nature (London), 402: 533-537].

Removal of BACE activity in mice by gene targeting completely abolishes Aβ production [see Luo, Y., et al., (2001) Nature Neuroscience, 4: 231-232; Roberds, S. L. et al., (2001) Human Molecular Genetics, 10: 1317-1324].

BACE −/− mice also show no detectable negative phenotypes, suggesting that disruption of BACE-mediated cleavage of APP does not produce additional undesired effects. This demonstrates that a drug substance capable of inhibiting β-secretase activity should lower or halt the synthesis of Aβ and should provide a safe treatment for Alzheimer's disease.

PCT Publications WO 92/14697, published Sep. 3, 1992, discloses substituted guanidines and derivatives thereof as modulators of neurotransmitter release.

At present there remains an urgent need to develop pharmaceutical agents capable for effective treatment in halting, slowing, preventing, and/or reversing the progression of Alzheimer's disease. Compounds that are effective inhibitors of beta-secretase, that inhibit beta-secretase mediated cleavage of APP, that are effective inhibitors of Aβ protein production by beta-secretase, and/or are effective in reducing soluble Aβ protein, amyloid beta deposits or amyloid beta plaques, are needed for effective treatment in halting, slowing, preventing, and/or reversing neurological disorders related to Aβ protein production, such as Alzheimer's disease.

SUMMARY OF THE DISCLOSURE

A series of indolyl acyl guanidine derivatives having the Formula (I)

or a stereoisomer; or a pharmaceutically acceptable salt thereof, wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ as defined below are effective inhibitors of the production of β-amyloid peptide (β-AP) from β-amyloid precursor protein (β-APP). The pharmacologic action of these compounds makes them useful for treating conditions responsive to the inhibition of β-AP in a patient; e.g., Alzheimer's Disease (AD) and Down's Syndrome. Therapy utilizing administration of these compounds or a pharmaceutical composition containing a therapeutically effective amount of at least one of these compounds to patients suffering from, or susceptible to, these conditions involves reducing β-AP available for accumulation and deposition in brains of these patients.

DETAILED DESCRIPTION

The present application comprises compounds of Formula I, their pharmaceutical formulations, and their use in inhibiting β-AP production in patients suffering from or susceptible to AD or other disorders resulting from β-AP accumulation in brain tissue. The compounds of Formula I which include stereoisomers and pharmaceutically acceptable salts thereof have the following formula and meanings:

wherein

-   R₁ is phenyl optionally substituted with one or more groups selected     from halogen, CN, CF₃, OH, —NH₂, —CH═CH-phenyl, pyridyl, thienyl,     C₃₋₆cycloalkyl, C₁₋₆alkoxy, C₂₋₆alkenyl, OR₂₁, C₁₋₆alkyl optionally     substituted with OH or —NH₂, —CH═CH—C₁₋₄alkyl optionally substituted     with C₁₋₄alkoxy or OH, phenyl optionally substituted with halogen,     —(CH₂)_(m)—NHC(═O)OC₁₋₆ alkyl optionally substituted with R₁₇,     —(CH₂)_(m)—NHC(═O)Ophenyl optionally substituted with halogen,     —(CH₂)_(m)—NHC(═O)R₁₈, —NH(C═O)R₁₉, —CH₂NH(SO₂)R₂₀, —CH₂morpholino     and 4-methylpiperazinyl; -   R₂ and R₃ are each independently hydrogen, methyl or hydroxymethyl; -   m is 0 or 1; -   R₄ and R₅ are each independently hydrogen or methyl; -   R₆ is hydrogen, C₁₋₆alkyl, C₁₋₆alkyl NHC(═O) or CN; -   R₇ is hydrogen, C₁₋₆ alkyl, or phenylmethyl in which said phenyl is     optionally substituted with one or more groups selected from     halogen, CN, CF₃, and OCH₃; -   R₈ is hydrogen, halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂,     benzyloxy or CF₃; -   R₁₇ is C₁₋₄alkoxy, halogen, C₂₋₃alkynyl, 4-nitrophenyl, benzyloxy or     benzothiophene 1,1-dioxide; -   R₁₈ is C₁₋₆alkyl or C₃₋₆cycloalkyl in which each are optionally     substituted with a group selected from halogen, CN, CF₃, —N(CH₃)₂,     C₁₋₄alkoxy, C₃₋₆cycloalkyl, morpholino, thienyl, imidazolyl,     pyridyl, azepinyl, benzothienyl, benzofuranyl, phenyl and     methoxyphenyl; -   R₁₉ is C₁₋₆alkyl, C₁₋₄alkoxy, C₃₋₆cycloalkyl optionally substituted     with —N(CH₃)₂ or methylthio, phenyl, pyridyl optionally substituted     with one or two halogen and C₁₋₄alkyl, benzothienyl,     1-methyl-2-pyrrolyl or furanyl; -   R₂₀ is C₁₋₆alkyl, 1,2-dimethyl-1H-imidazolyl, thienyl or phenyl in     which said phenyl is optionally substituted with a group selected     from acetyl, C₁₋₄alkoxy, benzyloxy, halogen, NH(C═O)CH₃ and nitro;     and -   R₂₁ is C₁₋₆alkyl optionally substituted with a group selected from     C₁₋₄alkoxy, halogen, phenyl, furanyl and methylamino;     or a nontoxic pharmaceutically acceptable salt thereof.

The present application also provides a method for the treatment or alleviation of disorders associated with β-amyloid peptide, especially Alzheimer's Disease, cerebral amyloid angiopathy and Down's Syndrome, which comprises administering together with a conventional adjuvant, carrier or diluent a therapeutically effective amount of a compound of Formula (I) or a nontoxic pharmaceutically acceptable salt thereof.

As used herein, the term “Aβ” denotes the protein designated Aβ, β-amyloid peptide, and sometimes β/A4, in the art. Aβ is an approximately 4.2 kilodalton (kD) protein of about 39 to 43 amino acids found in amyloid plaques, the walls of meningeal and parenchymal arterioles, small arteries, capillaries, and sometimes, venules. The isolation and sequence data for the first 28 amino acids are described in U.S. Pat. No. 4,666,829. The 43 amino acid sequence is well known in the art, see Colin Dingwall, Journal of Clinical Investigation, November 2001, 108 (9): 1243-1246; as well as PCT international patent application WO 01/92235, published Dec. 6, 2001, herein incorporated by reference in its entirety.

The term “APP”, as used herein, refers to the protein known in the art as β amyloid precursor protein. This protein is the precursor for Aβ and through the activity of “secretase” enzymes, as used herein, it is processed into Aβ. Differing secretase enzymes, known in the art, have been designated β secretase, generating the N-terminus of Aβ, α secretase cleaving around the 16/17 peptide bond in Aβ, and “γ secretases”, as used herein, generating C-terminal Aβ fragments ending at position 38, 39, 40, 42, and 43 or generating C-terminal extended precursors which are subsequently truncated to the above polypeptides.

The term “substituted,” as used herein and in the claims, means that any one or more hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency is not exceeded, and that the substitution results in a stable compound.

As used herein and in the claims, “alkyl” or “alkylene” is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms; for example, “C₁₋₆ alkyl” denotes alkyl having 1 to 6 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, pentyl, 3-methylbutyl, hexyl and the like. Preferred “alkyl” group, unless otherwise specified, is “C₁₋₄ alkyl”. Additionally, unless otherwise specified, “propyl” denotes n-propyl or i-propyl; “butyl” denotes n-butyl, i-butyl, sec-butyl, or t-butyl.

As used herein and in the claims, “alkenyl” or “alkenylene” is intended to include hydrocarbon chains of either a straight or branched configuration and one or more unsaturated carbon-carbon bonds which may occur in any stable point along the chain, for example, “C₂₋₆ alkenyl” include but are not limited to ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 3-methyl-2-butenyl, 2-pentenyl, 3-pentenyl, hexenyl, and the like.

As used herein and in the claims, “alkynyl” or “alkynylene” is intended to include hydrocarbon chains of either a straight or branched configuration and one or more carbon-carbon triple bonds which may occur in any stable point along the chain, for example, “C₂₋₆ alkynyl” include but not limited to ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, and the like.

“Alkoxy” or “alkyloxy” represents an alkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge. Examples of “C₁₋₆alkoxy” include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, and s-pentoxy. Preferred alkoxy groups are methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy.

As used herein and in the claims, “halogen” refers to fluoro, chloro, bromo, and iodo. Unless otherwise specified, preferred halogens are fluoro and chloro. “Counterion” is used to represent a small, negatively charged species such as chloride, bromide, hydroxide, acetate, sulfate, and the like.

“Cycloalkyl” is intended to include saturated ring groups, having the specified number of carbon atoms. For example, “C₃₋₆ cycloalkyl” denotes such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The compounds described herein may have asymmetric centers. It is understood, that whether a chiral center in an isomer is “R” or “S” depends on the chemical nature of the substituents of the chiral center. All configurations of compounds of the invention are considered part of the invention. Additionally, the carbon atom to which R₁ and R₂ is attached may describe a chiral carbon. Compounds of the present disclosure containing an asymmetrically substituted atom may be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials. Mixtures of isomers of the compounds of the examples or chiral precursors thereof can be separated into individual isomers according to methods which are known per se, e.g. fractional crystallization, adsorption chromatography or other suitable separation processes. Resulting racemates can be separated into antipodes in the usual manner after introduction of suitable salt-forming groupings, e.g. by forming a mixture of diastereosiomeric salts with optically active salt-forming agents, separating the mixture into diastereomeric salts and converting the separated salts into the free compounds. The enantiomeric forms may also be separated by fractionation through chiral high pressure liquid chromatography columns. Many geometric isomers of olefins and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and may be isolated as a mixture of isomers or as separated isomeric forms. All chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein and in the claims, “nontoxic pharmaceutically acceptable salts” refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.

The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, the disclosure of which is hereby incorporated by reference.

In the method of the present application, the term “therapeutically effective amount” means the total amount of each active component of the method that is sufficient to show a meaningful patient benefit, i.e., healing of acute conditions characterized by inhibition of β-amyloid peptide production. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. The terms “treat, treating, treatment” as used herein and in the claims means preventing or ameliorating diseases associated with β-amyloid peptide.

The compounds of the present application can be prepared in a number of ways well known to one skilled in the art of organic synthesis. The compounds of the present application can be synthesized using the methods described below, together with synthetic methods known in the art of synthetic organic chemistry, or variations thereon as appreciated by those skilled in the art. Preferred methods include, but are not limited to, those described below. All references cited herein are hereby incorporated in their entirety herein by reference.

The compounds may be prepared using the reactions and techniques described in this section. The reactions are performed in solvents appropriate to the reagents and materials employed and are suitable for the transformations being effected. Also, in the description of the synthetic methods described below, it is to be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment and workup procedures, are chosen to be the conditions standard for that reaction, which should be readily recognized by one skilled in the art. It is understood by one skilled in the art of organic synthesis that the functionality present on various portions of the molecule must be compatible with the reagents and reactions proposed. Such restrictions to the substituents which are compatible with the reaction conditions will be readily apparent to one skilled in the art and alternate methods must then be used.

Compounds of formula I are prepared as illustrated in Scheme 1. Treatment of commercially available PS-Wang-p-nitrophenyl carbonate resin (1) with S-methylisothiourea to afford solid-supported intermediate of formula 2. Intermediate of formula 2 is treated with intermediates of formula 3 and an amide-bond forming reagent (e.g. diisopropylcarbodiimide) to provide solid-supported intermediates of formula 4. Intermediates of formula 3 can be obtained commercially, can be prepared by methods known in the literature, or can be readily prepared by one skilled in the art. Intermediates of formula 4 are treated with intermediates of formula 5 to afford solid-supported intermediates of formula 6. Intermediates of formula 5 can be obtained commercially, can be prepared by methods known in the literature, or can be readily prepared by one skilled in the art. Treatment of intermediates of formula 6 with trifluoroacetic acid (TFA) will cleave the product from the solid support and provid compounds of formula I.

Alternatively, compounds of formula I are prepared as illustrated in Scheme 2. Intermediate of formula 7 is treated with intermediates of formula 3 and an amide-bond forming reagent (e.g. diisopropylcarbodiimide) to provide intermediates of formula 8. Intermediate of formula 7 can be prepared by analogy to methods known in the literature (See: Yuan, C.; Williams, R. M. Tetrahedron Lett. 1996, 37, 1945-1948). Intermediates of formula 3 can be obtained commercially, can be prepared by methods known in the literature, or can be readily prepared by one skilled in the art. Intermediates of formula 8 are treated with intermediates of formula 5 to afford intermediates of formula 9. Intermediates of formula 5 can be obtained commercially, can be prepared by methods known in the literature, or can be readily prepared by one skilled in the art. Treatment of intermediates of formula 9 with trifluoroacetic acid (TFA) will remove the BOC protecting group and provide compounds of formula I.

Compounds of formula 5 are prepared from intermediates of formula 10 as outlined in Scheme 3. Treatment of intermediates of formula 10 with N-bromosuccinimide in the presence of a radical initiator (AIBN or benzoylperoxide) can provide intermediates of formula 11. Intermediates of formula 10 can be obtained commercially, can be prepared by methods known in the literature, or can be readily prepared by one skilled in the art. Intermediates of formula 11 are converted to compounds of formula 5a via two routes. In the first route, intermediates of formula 11 are treated with sodium azide to provide intermediates of formula 12, which upon treatment with lithium aluminum hydride affords compounds of formula 5a. Alternatively, intermediates of formula 11 are treated with the potassium salt of phthalimide to provide intermediates of formula 13, which upon treatment with hydrazine affords compounds of formula 5a.

Compounds of formula 5b are also prepared from nitrile-containing intermediates of formula 14 as outlined in Scheme 4. Treatment of intermediates of formula 14 with strong hydride-based reducing agents such as lithium aluminum hydride or borane provides compounds of formula 5b.

Compounds of formula 5b are also prepared from carboxylic acid derivatives of formula 15 as outlined in Scheme 5. Treatment of intermediates of formula 15 with ammonia under typical amide bond formation conditions affords intermediates of formula 16, which upon treatment with strong hydride-based reducing agents such as borane provides compounds of formula 5b.

Bromo-substituted compounds of formula 17 are transformed into compounds of formula 19 as described in Scheme 6. Intermediates of formula 17 are treated with boronic acid intermediates of formula 18 and a palladium-based reagent to afford compounds of formula 19. Intermediates of formula 17 can be prepared via either of the synthetic routes outlined in Schemes 1 and 2. Intermediates of formula 18 can be obtained commercially, can be prepared by methods known in the literature, or can be readily prepared by one skilled in the art.

Bromo-substituted compounds of formula 20 were transformed into compounds of formula 22 as described in Scheme 7. Intermediates of formula 20 are treated with boronic acid intermediates of formula 21 and a palladium-based reagent to afford compounds of formula 22. Intermediates of formula 20 are prepared via either of the synthetic routes outlined in Schemes 1 and 2. Intermediates of formula 21 can be obtained commercially, can be prepared by methods known in the literature, or can be readily prepared by one skilled in the art.

Compounds of formula 26 can be prepared as described in Scheme 8. Solid-supported intermediates of formula 23 are treated with alcohols of formula 24 and the appropriate reagents for a Mitsunobu reaction to afford solid supported intermediates of formula 25. Intermediates of formula 23 can be prepared via the synthetic route outlined in Scheme 1. Alcohols of formula 24 can be obtained commercially, can be prepared by methods known in the literature, or can be readily prepared by one skilled in the art. Treatment of solid supported intermediates of formula 25 with TFA provides compounds of formula 26.

Compounds of formula 31, 32, and 33 can be prepared from a common solid supported intermediate as described in Scheme 9. Compounds of formula 31 are prepared by sequential treatment of solid supported intermediates of formula 27 with sulfonyl chloride intermediates of formula 28 and a base, then TFA. Intermediates of formula 27 can be prepared via the synthetic route outlined in Scheme 1. Sulfonyl chlorides of formula 28 can be obtained commercially, can be prepared by methods known in the literature, or can be readily prepared by one skilled in the art. Compounds of formula 33 are prepared by sequential treatment of solid supported intermediates of formula 27 with carboxylic acid intermediates of formula 30 and an amide bond forming reagent, then TFA. Intermediates of formula 27 can be prepared via the synthetic route outlined in Scheme 1. Carboxylic acids of formula 30 can be obtained commercially, can be prepared by methods known in the literature, or can be readily prepared by one skilled in the art. Compounds of formula 32 are prepared by sequential treatment of solid supported intermediates of formula 27 with chloroformate intermediates of formula 29 and a base, then TFA. Intermediates of formula 27 can be prepared via the synthetic route outlined in Scheme 1. Chloroformates of formula 29 can be obtained commercially, can be prepared by methods known in the literature, or can be readily prepared by one skilled in the art.

Compounds of formula 37 can be prepared as described in Scheme 10. Solid-supported intermediates of formula 34 are treated sequentially with p-nitrophenol chloroformate then amines of formula 35 to afford solid-supported intermediates of formula 36. Intermediates of formula 34 can be prepared via the synthetic route outlined in Scheme 1. Amines of formula 35 can be obtained commercially, can be prepared by methods known in the literature, or can be readily prepared by one skilled in the art. Treatment of solid supported intermediates of formula 36 with TFA provides compounds of formula 37.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The compounds of this application and their preparation can be understood further by the following working examples. These examples are meant to be illustrative of the present application, and are not to be taken as limiting thereof.

Chemical abbreviations used in the specification and Examples are defined as follows:

-   “Ac” for acetate, -   “APCI” for atmospheric pressure chemical ionization, -   “Boc” or “BOC” for t-butyloxycarbonyl, -   “BOP” for benzotriazol-1-yloxytris-(dimethylamino)-phosphonium     hexafluorophosphate, -   “Cbz” for benzyloxycarbonyl, -   “CDI” for 1,1′-carbonyldiimidazole, -   “CD₃OD” for deuteromethanol, -   “CDCl₃” for deuterochloroform, -   “DCC” for 1,3-dicyclohexylcarbodiimide, -   “DCM” for dichloromethane -   “DEAD” for diethyl azodicarboxylate, -   “DIEA”, “Hunig's base”, or “DIPEA” for N,N-diisopropylethylamine, -   “DMF” for N,N-dimethylformamide, -   “DMAP” for 4-dimethylaminopyridine, -   “DMPU” for 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidone, -   “DMSO” for dimethylsulfoxide, -   “DPPA” for diphenylphosphorylazide -   “EDC” or “EDCI” for 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide     hydrochloride, -   “Et” for ethyl, -   “EtOAC” for ethyl acetate, -   “HOAc” for acetic acid, -   “HOBt” for 1-hydroxybenzotriazole hydrate, -   “HATU” for O-(7-azabenzotriazol-1-yl)-N,N, N′,N′-tetramethyluronium     hexafluorophosphate, -   “HMPA” for hexamethylphosphoramide, -   “LDA” for lithium diisopropylamide, -   “LiHMDS” for lithium bis(trimethylsilyl)amide, -   “NaHMDS” for sodium bis(trimethylsilyl)amide, -   “NCS” for N-Chlorosuccinimide, -   “NMM” for 4-methylmorpholine, -   “PyBOP” for benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium     hexafluorophosphate, -   “TMSCH₂N₂” for (trimethylsilyl)diazomethane, -   “TMSN₃” for Azidotrimethylsilane, -   “TBTU” for O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium     tetrafluoroborate, -   “TEA” for triethylamine, -   “TFA” for trifluoroacetic acid, and -   “THF” for tetrahydrofuran.

Abbreviations used in the Examples are defined as follows: “° C.” for degrees Celsius, “MS” for mass spectrometry, “ESI” for electrospray ionization mass spectroscopy, “HR” for high resolution, “LC-MS” for liquid chromatography mass spectrometry, “eq” for equivalent or equivalents, “g” for gram or grams, “h” for hour or hours, “mg” for milligram or milligrams, “mL” for milliliter or milliliters, “mmol” for millimolar, “M” for molar, “min” for minute or minutes, “rt” for room temperature, “NMR” for nuclear magnetic resonance spectroscopy, “tlc” for thin layer chromatography, “atm” for atmosphere, and “α”, “β”, “R”, “S”, “E”, and “Z” are stereochemical designations familiar to one skilled in the art.

“HPLC” is an abbreviation used herein for high pressure liquid chromatography. Reverse-phase HPLC can be carried out using a Vydac C-18 column with gradient elution from 10% to 100% buffer B in buffer A (buffer A: water containing 0.1% trifluoroacetic acid, buffer B: 10% water, 90% acetonitrile containing 0.1% trifluoroacetic acid). If necessary, organic layers can be dried over sodium sulfate unless otherwise indicated. However, unless otherwise indicated, the following conditions are generally applicable. “LC-MS” refers to high pressure liquid chromatography carried out according to the definition for HPLC with a mass spectrometry detector.

Melting points were determined on a Mel-Temp II apparatus and are uncorrected. IR spectra were obtained on a single-beam Nicolet Nexus FT-IR spectrometer using 16 accumulations at a resolution of 4.00 cm-1 on samples prepared in a pressed disc of KBr or as a film on KBr plates. Proton NMR spectra (300 MHz, referenced to tetramethylsilane) were obtained on a Varian INOUA 300, Bruker Avance 300, Avance 400, or Avance 500 spectrometer. Data were referred to the lock solvent. Electrospray Ionization (ESI) experiments were performed on a Micromass II Platform single-quadrupole mass spectrometer, or on a Finnigan SSQ7000 mass spectrometer. HPLC analyses were obtained using a Rainin Dynamax C18 column with UV detection at 223 nm using a standard solvent gradient program as follows:

HPLC solvent conditions: When described as performed under “standard conditions”, Samples were dissolved in methanol (1 mg/mL) and run using the following gradient program with a solvent flow rate of 1.0 mL/min.

Acetonitrile (0.05% H₂O Time (min) TFA) (0.05% TFA) Initial 10 90 20.0 90 10 20–30 90 10

Preparatory HPLC: When described as performed under “standard conditions”, Samples (approx. 20 mg) were dissolved in methanol (10 mg/mL) and purified on a 25 mm×50 mm Vydac C18 colum with a 5 minute gradient elution from 10% to 100% buffer B in buffer A (buffer A: water containing 0.1% trifluoroacetic acid, buffer B: 10% water, 90% acetonitrile containing 0.1% trifluoroacetic acid) at 10 mL/minute.

Synthesis of Intermediates Preparation A 3,5-Dichloro-4-aminobenzylamine

Step A (1): To lithium aluminum hydride (0.57 g, 15 mmol) in dry THF (20 mL) was added dropwise 3,5-dichloro-4-aminobenzonitrile (1.87 g, 10 mmol) in THF (30 mL). The mixture was stirred at rt for 2 h. Then, sodium sulfate decahydrate (4.83 g, 15 mmol) was added and the mixture was stirred for 30 min. The solid was filtered off and washed with THF three times. The solvent was removed under vacuum and the residue was purified by chromatography with Methanol/DCM (3:7) as the eluant. 3,5-Dichloro-4-aminobenzylamine was obtained as an off-white solid (1.5 g, 80%). ¹H NMR (300 MHz, CD₃OD): δ3.65 (s, 2H), 7.2 (s, 2H).

Preparation B 4-acetamido-3,5-dichloro-benzylamine

Step B (1): To a solution of 3,5-dichloro-4-amino-benzonitrile (187 mg, 1 mmol) in 4 mL of THF at room temperature was added 2.2 mL of 1.0 M NaHMDS in THF. The resulting reaction mixture was stirred at room temperature for 30 min, at which time acetyl chloride (3.1 mmol) was added. DCM (100 mL) and water (100 mL) were added to the reaction mixture after being stirred overnight, followed by the addition of 5 mL of a 1.4 N HCl aqueous solution. The layers were separated and the aqueous layer was extracted with DCM (2×100 mL). The extracts were combined and solvents were evaporated in vacuo. The residue was purified by HPLC to give 4-acetamido-3,5-dichloro-benzonitrile. MS (ESI) (M−H)⁺=227.05. ¹H-NMR (300 MHz, CDCl₃) δ 7.63 (s, 2 H), 2.21 (s, 3 H).

Step B (2): To a solution of 4-acetamido-3,5-dichloro-benzonitrile (114 mg, 0.5 mmol) in 5 mL of THF was added lithium aluminum hydride (120 mg, 3.2 mmol) in one portion and the resulting mixture was stirred for 3 h. Sodium sulfate decahydrate (1 g, 3.2 mmol) was added in one portion and the mixture was stirred for 30 min. The solid was filtered off and the filtrate was concentrated. The residue was purified by HPLC to give 4-acetamido-3,5-dichloro-benzylamine (35 mg, 30%). ¹H NMR (500 MHz, CD₃OD): δ 7.46 (s, 2 H), 3.78 (s, 2H), 2.18 (s, 3 H).

Preparation C N-Boc-S-methylisothiourea

Step C (1): To a rapidly stirred suspension of S-Methylisothiourea hemisulfate (60.8 g, 0.437 mol) in CH₂Cl₂ (600 mL) was added 2N NaOH (300 mL, 0.6 mol). This mixture was cooled to 0° C. on an ice bath, and a solution of di-tert-butyl dicarbonate (43.2 g, 0.198 mol) was added dropwise over 6 h. Upon completion of the addition, the mixture was stirred an additional 20 min, diluted with 1 L of CH₂Cl₂ and the phases were separated. The organic portion was washed with water (2×500 ml) and dried over Na₂SO₄. Filtration and concentration provided the desired N-Boc-S-methylisothiourea as a white solid (35.5 g, 0.187 mol, 94% yield based on Boc₂O).

Preparation D tert-butyl N-4-acetamido-3,5-dichlorobenzylcarbamimidoylcarbamate

Step D (1): To a solution of mono-N-Boc-S-methylisothiourea (Preparation C, 764 mg, 4.0 mmol) and DIPEA (3.0 mL) in CH₂Cl₂ (8.0 mL) was added trifluoroacetic anhydride (924 mg, 4.4 mmol). The mixture was stirred at rt for 30 min, and then N-(4-(aminomethyl)-2,6-dichlorophenyl)acetamide (930 mg, 4.0 mmol) was added. The resulting mixture was stirred at rt overnight. The solvent was removed. Then 15 mL of MeOH and K₂CO₃ (1.2 g) were added. After stirring at rt for 30 min, the MeOH was removed. 50 mL of HCl (1 N) was added, and the mixture was washed with Et₂O (50 mL) and then neutralized with 50% NaOH to pH=12. The aqueous layer was then extracted with EtOAc (2×100 mL). The organic layer was dried and concentrated to give 1.1 g (yield 73%) of the title compound. MS (ESI) (M+H)⁺=375.06. ¹H-NMR (500 MHz, CD₃OD) δ 7.39 (s, 2 H), 4.41 (s, 2 H), 2.19 (s, 3 H), 1.45 (s, 9 H).

Preparation E 2-(6-bromo-1H-indol-3-yl)acetic acid

Step E (1): To a solution of 6-bromo-1H-indole (1.4 g, 7.14 mmol) in Et₂O (15.0 mL) was added oxalyl chloride (1.18 g, 9.29 mmol) dropwise at 0° C. under N₂. The reaction mixture was stirred at rt for 4 h. An orange precipitate formed, then 40 mL of saturated NaHCO₃ was added. The mixture was then stirred at 80° C. for 30 min. Et₂O was added to extract the unreacted starting materials. The resulting aqueous layer was acidified with concentrated HCl, and the product precipitated. The solid was filtered and dried to give 1.6 g (yield 83.6%) of 2-(6-bromo-1H-indol-3-yl)-2-oxoacetic acid. MS (ESI) (M−H)⁺=265.98. ¹H-NMR(500 MHz, CD₃OD) δ 8.39-8.78 (m, 1 H), 8.21 (d, J=8.24 Hz, 1 H), 7.69 (s, 1 H), 7.40 (d, J=8.55 Hz, 1 H).

Step E (2): To a solution of 2-(6-bromo-1H-indol-3-yl)-2-oxoacetic acid (780 mg, 2.9 mmol) from Step E (1) in MeOH/EtOH (20.0 mL/5.0 mL) was added 4-methylbenzenesulfonohydrazide (1.1 g, 5.8 mmol). The mixture was heated at reflux for 5 h. The solvent was removed. 15 mL of THF and NaBH₄ (658 mg, 17.4 mmol) were then added. The reaction mixture was stirred at 50° C. overnight and then at an additional 6 h at 80° C. The reaction mixture was diluted with H₂O and 1.0 N HCl and washed with EtOAc. The aqueous layer was concentrated and the residue was purified using reverse phase Prep-HPLC to give 18 mg of the title compound. MS (ESI) (M−H)⁺=251.98/254.01. ¹H-NMR (500 MHz, CD₃OD) δ 7.52 (s, 1 H), 7.46 (d, J=8.55 Hz, 1 H), 7.19 (s, 1 H), 7.14 (dd, J=8.55, 1.53 Hz, 1 H), 3.73 (s, 2 H).

Preparation F (3-chloro-4-(4-methylpiperazin-1-yl)phenyl)methanamine

Step F (1): To a solution of (4-(4-methylpiperazin-1-yl)phenyl)methanamine(204 mg, 1.0 mmol, Aldrich) in THF (2.0 mL) was added (Boc)₂O (327 mg, 1.5 mmol) and NaOH/H₂O (1.0 mL, 1 N). The mixture was stirred at rt for 1 h. EtOAc (100 mL) was added and the resulting solution was washed with H₂O (2×100 mL). The organic layer was dried and concentrated to give 250 mg (yield 82%) of tert-butyl 4-(4-methylpiperazin-1-yl)benzylcarbamate. LC-MS (M+H)⁺=306.35 ¹H-NMR(500 MHz, CD₃OD) δ 7.18 (d, J=8.55 Hz, 2 H), 6.94 (d, J=8.54 Hz, 2 H), 4.15 (s, 2 H), 3.16-3.22 (m, 4 H), 2.60-2.66 (m, 4 H), 2.36 (s, 3 H), 1.46 (s, 9 H).

Step F (2): To a solution of tert-butyl 4-(4-methylpiperazin-1-yl)benzylcarbamate (30 mg, 0.1 mmol) from step F (1) in CH₂Cl₂ (1.0 mL) was added NCS (14 mg, 0.1 mmol). The mixture was stirred at rt for 4 h, and then the mixture was concentrated to give tert-butyl 3-chloro-4-(4-methylpiperazin-1-yl)benzylcarbamate which was used without purification in the next step. LC-MS (M+H)⁺=340.29 ¹H-NMR (500 MHz, CD₃OD) δ 7.30 (s, 1 H), 7.18 (d, J=8.24 Hz, 1 H), 7.10 (d, J=8.24 Hz, 1 H), 4.16 (s, 2 H), 3.00-3.15 (m, 4 H), 2.62-2.81 (m, 7 H), 1.50(s, 9 H).

Step F (3): To a solution of tert-butyl 3-chloro-4-(4-methylpiperazin-1-yl)benzylcarbamate (36 mg, 0.1 mmol) from step F (2) in CH₂Cl₂ (0.5 mL) was added TFA (0.5 mmol). The mixture was stirred at rt for 2 h, and then the mixture was concentrated to give the title compound which was used without further purification. ¹H-NMR (500 MHz, CD₃OD) δ 7.59 (s, 1 H), 7.43 (d, J=8.24 Hz, 1 H), 7.27 (d, J=8.24 Hz, 1 H), 4.10 (s, 2 H), 3.60-3.70 (m, 2 H), 3.50-3.59 (m, 2 H), 3.29-3.42 (m, 2 H), 3.10-3.22 (m, 2 H), 2.98 (s, 3 H).

Preparation G 4-acetamido-3-chloro-5-methylbenzylamine

Step G (1). To a stirred solution of 4-amino-3-chloro-5-methylbenzonitrile (0.516 g, 3.1 mmol) in 10 mL of THF at rt was added 6.6 mL of 1.0 M NaHMDS in THF. The resulting reaction mixture was stirred at rt for 30 min., at which time acetyl chloride (3.1 mmol) was added. CH₂Cl₂ (100 mL) and water (100 mL) were added to the reaction mixture after being stirred overnight, followed by the addition of 5 mL of 1.4 N HCl aqueous solution. Layers were separated and the aqueous layer was extracted with CH₂Cl₂ (2×100 mL). The extracts were combined and solvents were evaporated in vacuo. The residue was purified by flash chromatography (5-20% EtOAc/DCM) to give 0.220 g of 4-acetamido-3-chloro-5-methylbenzonitrile. ¹H NMR (500 MHz, CDCl₃) δ 7.57 (s, 1 H), 7.46 (s, 1 H), 7.02 (s, 1 H), 2.31 (s, 3 H), 2.26 (s, 3 H).

Step G (2): 4-acetamido-3-chloro-5-methylbenzonitrile was reduced to 4-amino-3-chloro-5-methylbenzylamine by heating it at reflux with 2.2 eq. of LAH in THF for 2 hrs. ¹H NMR (500 MHz, CD₃OD) δ 7.31 (s, 1 H), 7.17 (s, 1 H), 3.73 (s, 2 H), 2.23 (s, 3 H), 2.15 (s, 3 H).

Preparation H 2-(5-cyano-1H-indol-3-yl)acetic acid

Step H (1): To a solution of 2-(5-bromo-1H-indol-3-yl)acetic acid (1.0 g 3.94 mmol) in MeOH (5 mL) and Et₂O (15 mL) was added (trimethylsilyl)diazomethane (2.0 M solution in hexanes, 10 mL). The reaction was stirred at rt for 2 h. The solvents were removed and the residue was purified by flash chromatography to yield 900 mg of methyl 2-(5-bromo-1H-indol-3-yl)acetate (yield 85%). ¹H NMR (500 MHz, CDCl₃) δ ppm 3.71 (s, 3 H) 3.72 (s, 2 H) 7.16 (d, J=2.14 Hz, 1 H) 7.21 (d, J=8.55 Hz, 1 H) 7.27 (dd, J=8.55, 1.83 Hz, 1 H) 7.72 (d, J=1.53 Hz, 1 H) 8.12 (s, 1 H)

Step H (2): To a solution of methyl 2-(5-bromo-1H-indol-3-yl)acetate (134 mg, 0.5 mmol) in NMP (1.0 mL) was added copper(I) cyanide (54 mg, 0.6 mmol). The reaction mixture was microwaved at 180° C. for 1.5 h. More copper (I) cyanide (54 mg, 0.6 mmol) was added, and the mixture was microwaved again at 180° C. for 2 h. 100 mL of EtOAc was added, and the reaction mixture was washed with 1.0 N HCl and H₂O. The organic layer was dried and the residue was purified by flash chromatography to give 50 mg of the methyl 2-(5-cyano-1H-indol-3-yl)acetate (yield 47%). MS (ESI) (M+H)⁺=215.16. ¹H NMR (500 MHz, CDCl₃) δ ppm 3.73 (s, 3 H) 3.77 (s, 2 H) 7.26 (d, J=2.14 Hz, 1 H) 7.37 (d, J=8.24 Hz, 1 H) 7.40 (dd, J=8.24, 1.22 Hz, 1 H) 7.95 (s, 1 H) 8.54 (s, 1 H).

Step H (3): To a solution of methyl 2-(5-cyano-1H-indol-3-yl)acetate (18 mg, 0.08 mmol) in THF (0.8 mL) was added a solution of LiOH (9.6 mg, 0.4 mmol) in water (0.3 mL). The mixture was stirred at rt for 3 h. 50 mL of EtOAc was added, and the resulting solution was washed with 0.1 N HCl and water. The organic layer was dried over Na₂SO₄, and the solvents was removed to give 11 mg of the title compound 2-(5-cyano-1H-indol-3-yl)acetic acid (yield, 68.7%). MS (ESI) (M−H)⁺=199.15. ¹H NMR (500 MHz, CD₃OD) δ ppm 4.86 (s, 2 H) 7.37 (s, 1 H) 7.40 (dd, J=8.24, 1.53 Hz, 1 H) 7.50 (d, J=8.54 Hz, 1 H) 8.01 (s, 1 H).

Preparation I 2-(5-bromo-2-(tert-butylcarbamoyl)-1H-indol-3-yl)acetic acid

Step I (I): To a solution of methyl 2-(5-bromo-1H-indol-3-yl)acetate ( 268 mg, 1.0 mmol) and tert-butyl isocyanate (149 mg, 1.5 mmol) in dichloromethane was added boron trifluoride diethyl etherate dropwise at −5° C. under nitrogen. The mixture was stirred from −5° C. to rt and at rt overnight. 100 mL of EtOAc was added, and the resulting solution was washed with 0.1 N HCl and water. The organic layer was dried over Na₂SO₄, and the solvents was removed to give 340 mg of the crude methyl 2-(5-bromo-2-(tert-butylcarbamoyl)-1H-indol-3-yl)acetate (yield 92.6%). MS (ESI) (M+H)⁺=369.05. ¹H NMR (500 MHz, CDCl₃) δ ppm 1.51 (s, 9 H) 3.74 (s, 3 H) 3.86 (s, 2 H) 7.26 (d, J=8.55 Hz, 1 H) 7.35 (dd, J=8.55, 1.83 Hz, 1 H) 7.79 (d, J=1.53 Hz, 1 H) 8.01 (s, 1 H) 9.17 (s, 1 H).

Step I (2): To a solution of methyl 2-(5-bromo-2-(tert-butylcarbamoyl)-1H-indol-3-yl)acetate (80 mg, 0.22 mmol) in THF (2.0 mL) was added a solution of LiOH (16 mg, 0.66 mmol) in water (0.5 mL). The mixture was stirred at rt for 3 h. 100 mL of EtOAc was added, and the resulting solution was washed with 0.1 N HCl and water. The organic layer was dried over Na₂SO₄, and the solvents was removed to give the title compound 2-(5-bromo-2-(tert-butylcarbamoyl)-1H-indol-3-yl)acetic acid which was used without further purification. MS (ESI) (M+H)⁺=355.03.

Preparation J 2-(5-bromo-2-(tert-butylcarbamoyl)-1H-indol-3-yl)acetic acid

Step J (1): To a solution of methyl 2-(5-bromo-2-(tert-butylcarbamoyl)-1H-indol-3-yl)acetate (124 mg, 0.34 mmol) in toluene was added POCl₃ (182 mg, 1.19 mmol). The mixture was stirred at 80° C. for 6 h. The solvent was removed. The product was isolated by flash chromatography to give 50 mg of methyl 2-(5-bromo-2-cyano-1H-indol-3-yl)acetate (yield 50.2%). MS (ESI) (M+H)⁺=295.03. ¹H NMR (500 MHz, CDCl₃) δ ppm 3.77 (s, 3 H) 3.89 (s, 2 H) 7.11 (d, J=8.85 Hz, 1 H) 7.36 (dd, J=8.85, 1.83 Hz, 1 H) 7.72 (d, J=1.83 Hz, 1 H) 9.00 (s, 1 H).

Step J (2): To a solution of methyl 2-(5-bromo-2-cyano-1H-indol-3-yl)acetate (50 mg, 0.17 mmol) in THF (1.0 mL) was added a solution of LiOH (12 mg, 0.5 mmol) in water (0.5 mL). The mixture was stirred at rt for 3 h. 100 mL of EtOAc was added, and the resulting solution was washed with 0.1 N HCl and water. The organic layer was dried over Na₂SO₄, and the solvents was removed to give 45 mg of the title compound 2-(5-bromo-2-cyano-1H-indol-3-yl)acetic acid (yield 94.7%) which was used without further purification. ¹H NMR (500 MHz, CDCl₃) δ ppm 3.89 (s, 2 H) 7.34 (d, J=8.85 Hz, 1 H) 7.43 (dd, J=8.85, 1.83 Hz, 1 H) 7.83 (d, J=1.53 Hz, 1 H).

EXAMPLE 1 N-(4-{N′-[2-(6-Bromo-1H-indol-3-yl)-acetyl]-guanidinomethyl}-2,6-dichloro-phenyl)-acetamide

Step 1 (A): To a solution of 2-(6-bromo-1H-indol-3-yl)acetic acid (preparation E, 18 mg, 0.07 mmol) in CH₂Cl₂ (2.0 mL) was added HATU (32 mg, 0.084 mmol) and DIPEA (0.5 mL). The mixture was stirred at rt for 15 min, and then tert-butyl N-4-acetamido-3,5-dichlorobenzylcarbaniimidoylcarbamate (preparation D, 31 mg, 0.084 mmol) was added. The reaction mixture was stirred at rt overnight. The resulting crude product was purified by silica gel chromatography to give 18 mg of tert-butyl N-(4-acetamido-3,5-dichlorobenzyl)-N′-(2-(6-bromo-1H-indol-3-yl)acetyl)carbamimidoylcarbamate. MS (ESI) (M+H)⁺=610.03/611.99/613.96.

Step 1 (B): To a solution of tert-butyl N-(4-acetamido-3,5-dichlorobenzyl)-N′-(2-(6-bromo-1H-indol-3-yl)acetyl)carbamimidoylcarbamate (12 mg, 0.020 mmol) from step 1 (A) in CH₂Cl₂ (0.5 mL) was added TFA (0.5 mL). The mixture was stirred for 2 h at rt. The solvent was removed and the residue was purified with reverse phase Prep-HPLC to give 5 mg of the title compound. MS (ESD (M+H)⁺=509.91/511.89/513.91. ¹H-NMR(500 MHz, CD₃OD) δ 7.57 (s, 1 H), 7.46-7.51 (m, 1 H), 7.42-7.47 (m, 2 H), 7.31 (s, 1 H), 7.18 (dd, J=8.55, 1.83 Hz, 1 H), 4.54 (s, 2 H), 3.97 (s, 2 H), 2.19 (s, 3 H).

EXAMPLE 2 N-(2,6-Dichloro-4-{N′-[2-(5-methoxy-1H-indol-3-yl)-acetyl]-guanidinomethyl}-phenyl)-acetamide

Step 2 (A): To a mixture of 2-(5-methoxy-1H-indol-3-yl)acetic acid (205 mg, 1.0 mmol)) and CH₂Cl₂/CH₃CN/DIPEA (4.0 mL/0.5 mL/0.5 mL) was added HATU (418 mg, 1.1 mmol). The mixture was stirred at rt for 5 min, and then N-Boc-S-methylisothiourea (preparation C, 190 mg, 1.0 mmol) was added. The reaction was stirred at rt overnight. The resulting crude product was purified by silica gel chromatography to give 280 mg (yield 74%) of tert-butyl N-Boc-S-methylisothiourea-(2-(5-methoxy-1H-indol-3-yl)acetyl)(methylthio)-carbonoimidoylcarbamate. MS (ESI) (M+H)⁺=378.08. ¹H-NMR (500 MHz, CDCl₃) δ 7.22 (d, J=8.85 Hz, 1 H), 7.13 (s, 1 H), 6.92-6.99 (m, 1 H), 6.85 (dd, J=8.70, 2.29 Hz, 1 H), 3.83 (s, 5 H), 2.31 (s, 3 H), 1.26-1.51 (m, 9 H).

Step 2 (B): To a solution of tert-butyl N-(2-(5-methoxy-1H-indol-3-yl)acetyl) (methylthio)carbonoimidoylcarbamate (50 mg, 0.13 mmol) from step 2 (A) in CH₂Cl₂ (1.0 mL) was added N-(4-(aminomethyl)-2,6-dichlorophenyl)acetamide (47 mg, 0.2 mmol) and DIPEA (0.5 mL). The reaction was stirred at rt overnight. The resulting crude product was purified by silica gel chromatography to give 45 mg (yield 62%) of tert-butyl N-(4-acetamido-3,5-dichlorobenzyl)-N-(2-(5-methoxy-1H-indol-3-yl)acetyl)carbamimidoylcarbamate. LC-MS (M+H)⁺=562.31. ¹H-NMR (500 MHz, CD₃OD) δ 7.37 (s, 2 H), 7.22-7.30 (m, 2 H), 7.00 (d, J=2.44 Hz, 1 H), 6.79 (dd, J=8.70, 2.29 Hz, 1 H), 4.44-4.51 (m, 2 H), 3.87 (s, 2 H), 3.74-3.82 (m, 3 H), 2.17 (s, 3 H), 1.23-1.55 (m, 9 H).

Step 2 (C): To a solution of tert-butyl N-(4-acetamido-3,5-dichlorobenzyl)-N′-(2-(5-methoxy-1H-indol-3-yl)acetyl)carbamimidoylcarbamate (45 mg, 0.08 mmol) from step 2 (B) in CH₂Cl₂ (2.0 mL) was added TFA (1.0 mL). The mixture was stirred for 3 h at rt. The solvent was removed and the residue was purified with reversed phase Prep-HPLC to give 25 mg of the title compound. MS (ESI) (M+H)⁺=462.05. ¹H-NMR (300 MHz, CD₃OD) δ 7.44 (s, 2 H), 7.27 (d, J=5.12 Hz, 2 H), 7.04 (d, J=2.56 Hz, 1 H), 6.83 (dd, J=8.78, 2.20 Hz, 1 H), 4.52 (s, 2 H), 3.95 (s, 2 H), 3.83 (s, 3 H), 2.19 (s, 3 H).

EXAMPLE 3 N-(3,5-Dichloro-benzyl)-N′-[2-(5-methoxy-1H-indol-3-yl)-acetyl]-guanidine

In a manner similar to that used in the preparation of the compound of example 2, but using dichlorobenzyl amine in step 3 (B), the title compound was prepared. MS (ESI) (M+H)⁺=405.28. ¹H-NMR (500 MHz, CDCl₃) δ 8.25 (d, 1H), 7.64 (s, 1 H), 7.53 (s, 1 H), 7.09 (s, 1 H), 6.88 (s, 2 H), 6.84 (bs, 4 H), 6.62 (d, 1 H), 4.37 (s, 2 H), 4.13 (s, 2 H), 3.89 (s, 3 H).

EXAMPLE 4 N-(3,5-Dichloro-benzyl)-N′-[2-(5-fluoro-1H-indol-3-yl)-acetyl]-guanidine

In a manner similar to that used in the preparation of the compound of example 2, but using 2-(5-fluoro-1H-indol-3-yl)acetic acid in step 4 (A) and dichlorobenzyl amine in step 4 (B), the title compound was prepared. MS (ESI) (M+H)+=393.24. ¹H-NMR (500 MHz, CDCl₃) δ 7.53 (s, 1 H), 7.08 (d, 1 H), 6.87 (bs, 3 H), 6.84 (bs, 4 H), 6.42 (m, 1 H), 4.38 (s, 2 H), 4.12 (s, 2 H).

EXAMPLE 5 N-((4-amino-3,5-dichlorobenzylamino)(amino)methylene)-2-(1-methyl-1H-indol-3-yl)acetamide

In a manner similar to that used in the preparation of the compound of example 2, but using 2-(1-methyl-1H-indol-3-yl)acetic acid in step 5 (A) and 3,5-dichloro-4-aminobenzylamine (preparation A) in step 5 (B), the title compound was prepared. MS (ESI) (M+H)⁺=404.07 ¹H-NMR(500 MHz, CD₃OD) δ 7.54 (d, J=7.93 Hz, 1 H), 7.40 (d, J=8.24 Hz, 1 H), 7.15-7.32 (m, 4 H), 7.04-7.15 (m, 1 H), 4.33 (s, 2 H), 3.95 (s, 2 H), 3.80 (s, 3 H).

EXAMPLE 6 N-((4-amino-3,5-dichlorobenzylamino)(amino)methylene)-2-(1-(4-fluorobenzyl)-1H-indol-3-yl)acetamide

In a manner similar to that used in the preparation of the compound of example 2, but using 2-(1-(4-fluorobenzyl)-1H-indol-3-yl)acetic acid in step 6 (A) and 3,5-dichloro-4-aminobenzylamine (preparation A) in step 6 (B), the title compound was prepared. MS (ESI) (M+H)⁺=498.08 ¹H-NMR(500 MHz, CD₃OD) δ 7.58 (d, J=7.63 Hz, 1 H), 7.26-7.40 (m, 2 H), 7.13-7.25 (m, 5 H), 7.05-7.13 (m, 1 H), 7.01 (t, J=8.70 Hz, 2 H), 5.33 (s, 2 H), 4.32 (s, 2 H), 3.96 (s, 2 H).

EXAMPLE 7 N-((4-amino-3,5-dichlorobenzylamino)(amino)methylene)-2-(5-methoxy-1H-indol-3-yl)acetamide

In a manner similar to that used in the preparation of the compound of example 2, but using 2-(5-methoxy-1H-indol-3-yl)acetic acid in step 7 (A) and 3,5-dichloro-4-aminobenzylamine (preparation A) in step 7 (B), the title compound was prepared. MS (ESI) (M+H)⁺=419.99 ¹H-NMR (500 MHz, CD₃OD) δ 7.29 (d, J=8.85 Hz, 1 H), 7.25 (s, 1 H), 7.21 (s, 2 H), 7.03 (d, J=2.44 Hz, 1 H), 6.82 (dd, J=8.85, 2.44 Hz, 1 H), 4.33 (s, 2 H), 3.92 (s, 2 H), 3.82 (s, 3 H).

EXAMPLE 8 N-((4-amino-3,5-dichlorobenzyl amino)(amino)methylene)-2-(1H-indol-3-yl)propanamide

In a manner similar to that used in the preparation of the compound of example 2, but using 2-(1H-indol-3-yl)propionic acid in step 8 (A) and 3,5-dichloro-4-aminobenzylamine (preparation A) in step 8 (B), the title compound was prepared. MS (ESI) (M+H)⁺=404.03 ¹H-NMR (500 MHz, CD₃OD) δ 7.55 (d, J=7.93 Hz, 1 H), 7.40 (d, J=7.93 Hz, 1 H), 7.28 (s, 1 H), 7.19 (s, 2 H), 7.12-7.17 (m, 1 H), 7.02-7.08 (m, 1 H), 4.31 (s, 2 H), 4.14 (m, 1 H), 1.63 (d, J=7.02 Hz, 3 H).

EXAMPLE 9 N-(2-Chloro-4-{N′-[2-(5-methoxy-1H-indol-3-yl)-acetyl]-guanidinomethyl}-6-methyl-phenyl)-acetamide

In a manner similar to that used in the preparation of the compound of example 2, but using 2-(5-methoxy-1H-indol-3-yl)acetic acid in step 9 (A) and 4-amino-3-chloro-5-methylbenzylamine (preparation G) in step 9 (B), the title compound was prepared. MS (ESI) (M+H)⁺=442.04 ¹H-NMR (500 MHz, CD₃OD) δ 7.20-7.39 (m, 3 H), 7.16 (s, 1 H), 7.05 (d, J=2.14 Hz, 1 H), 6.82 (dd, J=8.55, 2.44 Hz, 1 H), 4.46 (s, 2 H), 3.93 (s, 2 H), 3.82 (s, 3 H), 2.24 (s, 3 H), 2.18 (s, 3 H).

EXAMPLE 10 N-(2,6-Dichloro-4-{N′-[2-(5-methoxy-2-methyl-1H-indol-3-yl)-acetyl]-guanidinomethyl}-phenyl)-acetamide

In a manner similar to that used in the preparation of the compound of example 2, but using 2-(5-methoxy-2-methyl-1H-indol-3-yl)acetic acid in step 10 (A) and 4-acetamido-3,5-dichloro-benzylamine (preparation B) in step 10 (B), the title compound was prepared. MS (ESI) (M+H)⁺=476.05 ¹H-NMR (300 MHz, CD₃OD) δ 7.42 (s, 2 H), 7.18 (d, J=8.42 Hz, 1 H), 6.96 (d, J=2.56 Hz, 1 H), 6.64-6.84 (m, J=2.56 Hz, 1 H), 4.48 (s, 2 H), 3.88 (s, 2 H), 3.80 (s, 3 H), 2.40 (s, 3 H), 2.19 (s, 3 H).

EXAMPLE 11 N-(2,6-Dichloro-4-{N′-[2-(2-methyl-1H-indol-3-yl)-acetyl]-guanidinomethyl}-phenyl)-acetamide

In a manner similar to that used in the preparation of the compound of example 2, but using 2-(2-methyl-1H-indol-3-yl)acetic acid in step 11 (A) and 4-acetamido-3,5-dichloro-benzylamine (preparation B) in step 11 (B), the title compound was prepared. MS (ESI) (M+H)⁺=446.08 ¹H-NMR (500 MHz, CD₃OD) δ 7.36-7.50 (m, 3 H), 7.30 (d, J=7.93 Hz, 1 H), 7.00-7.10 (m, 2 H), 4.52 (s, 2 H), 3.92 (s, 2 H), 2.44 (s, 3 H), 2.19 (s, 3 H).

EXAMPLE 12 N-(2,6-Dichloro-4-{N′-[2-(5-fluoro-1H-indol-3-yl)-acetyl]-guanidinomethyl}-phenyl)-acetamide

In a manner similar to that used in the preparation of the compound of example 2, but using 2-(5-fluoro-1H-indol-3-yl)acetic acid in step 12 (A) and 4-acetamido-3,5-dichloro-benzylamine (preparation B) in step 12 (B), the title compound was prepared. MS (ESI) (M+H)⁺=449.95 ¹H-NMR (500 MHz, CD₃OD) δ 7.45 (s, 2 H), 7.32-7.41 (m, 2 H), 7.24 (dd, J=9.77, 2.44 Hz, 1 H), 6.84-7.02 (m, 1 H), 4.54 (s, 2 H), 3.95 (s, 2 H), 2.19 (s, 3 H).

EXAMPLE 13 N-(4-{N′-[2-(5-Benzyloxy-1H-indol-3-yl)-acetyl]-guanidinomethyl}-2,6-dichloro-phenyl)-acetamide

In a manner similar to that used in the preparation of the compound of example 2, but using 2-(5-benzyloxy-1H-indol-3-yl)acetic acid in step 13 (A) and 4-acetamido-3,5-dichloro-benzylamine (preparation B) in step 13 (B), the title compound was prepared. MS (ESI) (M+H)⁺=538.03 ¹H-NMR (500 MHz, CD₃OD) δ 7.18-7.57 (m, 9 H), 7.11 (d, J=2.14 Hz, 1 H), 6.92 (dd, J=8.85, 2.44 Hz, 1 H), 5.11 (s, 2 H), 4.53 (s, 2 H), 3.94 (s, 2 H), 2.19 (s, 3 H).

EXAMPLE 14 N-(2,6-Dichloro-4-{N′-[2-(5-hydroxy-1H-indol-3-yl)-acetyl]-guanidinomethyl}-phenyl)-acetamide

In a manner similar to that used in the preparation of the compound of example 2, but using 2-(5-hydroxy-1H-indol-3-yl)acetic acid in step 14 (A) and 4-acetamido-3,5-dichloro-benzylamine (preparation B) in step 14 (B), the title compound was prepared. MS (ESI) (M+H)⁺=448.05 ¹H-NMR (500 MHz, CD₃OD) δ 7.41 (s, 2 H), 7.15-7.30 (m, 2 H), 6.93 (d, J=2.44 Hz, 1 H), 6.73 (dd, J=8.70, 2.29 Hz, 1 H), 4.47 (s, 2 H), 3.88 (s, 2 H), 2.19 (s, 3 H).

EXAMPLE 15 N-(4-{N′-[2-(6-Benzyloxy-1H-indol-3-yl)-acetyl]-guanidinomethyl}-2,6-dichloro-phenyl)-acetamide

In a manner similar to that used in the preparation of the compound of example 2, but using 2-(6-benzyloxy-1H-indol-3-yl)acetic acid in step 15 (A) and 4-acetamido-3,5-dichloro-benzylamine (preparation B) in step 15 (B), the title compound was prepared. MS (ESI) (M+H)⁺=538.07 ¹H-NMR(500 MHz, CD₃OD) δ 7.34-7.50 (m, 7 H), 7.24-7.34 (m, 1 H), 7.17 (s, 1 H), 7.00 (d, J=2.14 Hz, 1 H), 6.83 (dd, J=8.70, 2.29 Hz, 1 H), 5.10 (s, 2 H), 4.50 (s, 2 H), 3.92 (s, 2 H), 2.19 (s, 3 H).

EXAMPLE 16 N-(2,6-Dichloro-4-{N′-[2-(6-fluoro-1H-indol-3-yl)-acetyl]-guanidinomethyl}-phenyl)-acetamide

In a manner similar to that used in the preparation of the compound of example 2, but using 2-(6-fluoro-1H-indol-3-yl)acetic acid in step 16 (A) and 4-acetamido-3,5-dichloro-benzylamine (preparation B) in step 16 (B), the title compound was prepared. MS (ESI) (M+H)⁺=450.08 ¹H-NMR(500 MHz, CD₃OD) δ 7.52 (dd, J=8.55, 5.19 Hz, 1 H), 7.42 (s, 2 H), 7.28 (s, 1 H), 7.09 (dd, J=9.77, 2.14 Hz, 1 H), 6.77-6.95 (m, 1 H), 4.50 (s, 2 H), 3.95 (s, 2 H), 2.19 (s, 3 H).

EXAMPLE 17 N-(4-Amino-3,5-dichloro-benzyl)-N′-[2-(5-bromo-1H-indol-3-yl)-acetyl]-guanidine

In a manner similar to that used in the preparation of the compound of example 2, but using 2-(5-bromo-1H-indol-3-yl)acetic acid in step 17 (A) and 3,5-Dichloro-4-aminobenzylamine (preparation A) in step 17 (B), the title compound was prepared. MS (ESI) (M+H)⁺=467.90/469.86/471.83 ¹H-NMR (500 MHz, CD₃OD) δ 7.71 (s, 1 H), 7.32 (m, 2 H), 7.16-7.28 (m, 3 H), 4.35 (s, 2 H), 3.93 (s, 2 H).

EXAMPLE 18 N-[2-(5-Bromo-1H-indol-3-yl)-acetyl]-N′-[4-(4-methyl-piperazin-1-yl)-benzyl]-guanidine

In a manner similar to that used in the preparation of the compound of example 2, but using 2-(5-bromo-1H-indol-3-yl)acetic acid in step 18 (A) and (4-(4-methylpiperazin-1-yl)phenyl)methanamine in step 18 (B), the title compound was prepared. MS (ESI) (M+H)⁺=483.08/485.08 ¹H-NMR (500 MHz, CD₃OD) δ 7.72 (d, J=1.22 Hz, 1 H), 7.17-7.38 (m, 5 H), 7.03 (d, J=8.55 Hz, 2 H), 4.43 (s, 2 H), 3.92 (s, 2 H), 3.76-3.88 (m, 2 H), 3.53-3.66 (m, 2 H), 3.17-3.29 (m, 2 H), 2.99-3.15 (m, 2 H), 2.96 (s, 3 H).

EXAMPLE 19 N-[2-(5-Bromo-1H-indol-3-yl)-acetyl]-N′-[3-chloro-4-(4-methyl-piperazin-1-yl)-benzyl]-guanidine

In a manner similar to that used in the preparation of the compound of example 2, but using 2-(5-bromo-1H-indol-3-yl)acetic acid in step 19 (A) and (3-chloro-4-(4-methylpiperazin-1-yl)phenyl)methanamine (preparation F) in step 19 (B), the title compound was prepared. MS (ESI) (M+H)⁺=517.02/518.98 ¹H-NMR (500 MHz, CD₃OD) δ 7.73 (s, 1 H), 7.45 (s, 1 H), 7.27-7.38 (m, 3 H), 7.17-7.26 (m, 2 H), 4.49 (s, 2 H), 3.93 (s, 2 H), 3.57-3.68 (m, 2 H), 3.47-3.58 (m, 2 H), 3.28-3.40 (m, 2 H), 3.06-3.18 (m, 2 H), 2.96-3.01 (s, 3 H).

EXAMPLE 20 N-(4-{N′-[2-(5-Bromo-1H-indol-3-yl)-acetyl]-guanidinomethyl}-2,6-dichloro-phenyl)-acetamide

In a manner similar to that used in the preparation of the compound of example 1, but using 2-(5-bromo-1H-indol-3-yl)acetic acid in step 20 (A), the title compound was prepared. MS (ESI) (M+H)⁺=509.84/511.92/513.90 ¹H-NMR (500 MHz, CD₃OD) δ 7.73 (d, J=1.53 Hz, 1 H), 7.46 (s, 2 H), 7.29-7.40 (m, 2 H), 7.26 (d, J=1.83 Hz, 1 H), 4.54 (s, 2 H), 3.96 (s, 2 H), 2.19 (s, 3 H).

EXAMPLE 21 N-(2,6-Dichloro-4-{(′-[2-(5-fluoro-2-methyl-1H-indol-3-yl)-acetyl]-guanidinomethyl}-phenyl)-acetamide

In a manner similar to that used in the preparation of the compound of example 1, but using 2-(5-fluoro-2-methyl-1H-indol-3-yl)acetic acid in step 21 (A), the title compound was prepared. MS (ESI) (M+H)⁺=464.09 ¹H-NMR (500 MHz, CD₃OD) δ 7.45 (s, 2 H), 7.24 (dd, J=8.70, 4.43 Hz, 1 H), 7.12 (dd, J=9.77, 2.44 Hz, 1 H), 6.74-6.92 (m, 1 H), 4.53 (s, 2 H), 3.89 (s, 2 H), 2.43 (s, 3 H), 2.19 (s, 3 H).

EXAMPLE 22 N-(2,6-Dichloro-4-{N′-[2-(7-fluoro-2-methyl-1H-indol-3-yl)-acetyl]-guanidinomethyl}-phenyl)-acetamide

In a manner similar to that used in the preparation of the compound of example 1, but using 2-(7-fluoro-2-methyl-1H-indol-3-yl)acetic acid in step 22 (A), the title compound was prepared. MS (ESI) (M+H)+=464.00 1H-NMR(500 MHz, CD₃OD) δ 7.44 (s, 2 H), 7.24 (d, J=7.93 Hz, 1 H), 6.90-7.01 (m, 1 H), 6.72-6.89 (m, 1 H), 4.52 (s, 2 H), 3.93 (s, 2 H), 2.45 (s, 3 H), 2.19 (s, 3 H).

EXAMPLE 23 N-(4-{N′-[2-(5-Cyano-1H-indol-3-yl)-acetyl]-guanidinomethyl}-2,6-dichloro-phenyl)-acetamide

In a manner similar to that used in the preparation of the compound of example 1, but using 2-(5-cyano-1H-indol-3-yl)acetic acid (preparation H) in step 23 (A), the title compound was prepared. ¹H-NMR (500 MHz, CD₃OD) δ ppm 2.19 (s, 3 H) 4.03 (s, 2 H) 4.56 (s, 2 H) 7.45 (m, 4 H) 7.55 (d, J=8.55 Hz, 1 H) 8.06 (s, 1 H)

EXAMPLE 24 3-{2-[N′-(4-Acetylamino-3,5-dichloro-benzyl)-guanidino]-2-oxo-ethyl}-5-bromo-1H-indole-2-carboxylic acid tert-butylamide

In a manner similar to that used in the preparation of the compound of example 1, but using 2-(5-bromo-2-(tert-butylcarbamoyl)-1H-indol-3-yl)acetic acid (preparation I) in step 24 (A), the title compound was prepared. ¹H-NMR (500 MHz, CD₃OD) δ ppm 1.51 (s, 9 H) 2.20 (s, 3 H) 4.13 (s, 2 H) 4.54 (s, 2 H) 7.37 (m, 4 H) 7.84 (s, 1 H).

EXAMPLE 25 N-(4-{N′-[2-(5-Bromo-2-cyano-1H-indol-3-yl)-acetyl]-guanidinomethyl}-2,6-dichloro-phenyl)-acetamide

In a manner similar to that used in the preparation of the compound of example 1, but using 2-(5-bromo-2-(tert-butylcarbamoyl)-1H-indol-3-yl)acetic acid (preparation J) in step 25 (A), the title compound was prepared. ¹H-NMR (500 MHz, CD₃OD) δ ppm 2.19 (d, J=12.82 Hz, 3 H) 3.79 (s, 2 H) 4.31 (s, 2 H) 7.00 (s, 1 H) 7.26 (s, 1 H) 7.35 (d, J=8.24 Hz, 1 H) 7.85 (s, 1 H).

Biological Methods

There are a number of methods by which inhibitors of the BACE enzyme can be identified experimentally. The enzyme can be obtained from membrane samples from natural tissues or cultured cells or can be expressed recombinantly in a host cell by well known methods of molecular biology. The whole enzyme or a portion thereof can be expressed, for example, in bacterial, insect or mammalian cells to obtain a catalytically active enzyme species. The enzymatic activity and/or ligand binding capability of the enzyme can be assessed within these membrane samples, or the enzyme can be purified to varying extents. As an illustrative example, the nucleic acid sequence encoding the pro and catalytic domains of human BACE can be appended on the 5′ end with an untranslated and signal sequence from the gene for acetylcholinesterase, and on the 3′ end with a sequence encoding a poly-histidine tag. This cDNA can then be expressed in Drosophila melanogaster S2 cells in which the signal and pro sequences of the transcribed/translated protein are removed by cellular proteases and the catalytic domain, appended by a C-terminal poly-histidine tag, is secreted out into the cellular medium. The enzyme can then be purified from the culture medium by nickel affinity chromatography by methods well known to those trained in the art [Mallender, W. et al., (2001) “Characterization of recombinant, soluble beta-secretase from an insect cell expression system.” Mol. Pharmacol. 59: 619-626]. Similar strategies for expressing and purifying various forms of BACE in bacterial, mammalian and other cell types would be known to one skilled in the art. A preferred method for determining the potency of a test compound in binding to the BACE enzyme is by moitoring the displacement of a suitable radioligand.

Radioligand displacement assays with a radiolabeled BACE inhibitor (WO 2004 013098, compound 3, where the methoxy group is substituted for C(³H)₃) were carried out using standard methods (Keen, M. (1999) in Receptor Binding Techniques (Walker, J. M. ed) p. 106 Humana Press, Totowa, N.J.). The HEK293-9B.A1 cell line, which overexpresses the BACE1 enzyme, was derived from HEK293 cells (Simmons, N. L. (1990) A cultured human renal epithelioid cell line responsive to vasoactive intestinal peptide. Exp. Physiol. 75:309-19.) by RAGE™ (Harrington, J. J. et al. (2001) Creation of genome-wide protein expression libraries using random activation of gene expression. Nat. Biotechnol. 19:440-5; U.S. Pat. No. 6,410,266 and 6,361,972). T225 flask cultures of HEK293-9B.A1 were grown to 80% confluency in DMEM supplemented with 2 mM L-glutamine, 10 μg/ml penecillin, 10 μg/ml streptomycin, 3 μg/ml puromycin, 100 nM methotrexate, and 10% fetal bovine serun (Invitrogen, Carlsbad, Calif.), harvested, and resuspended at 2×10⁸ cells per 10 ml of lysis buffer consisting of 50 mM HEPES pH 7.0 containing a protease inhibitor cocktail of AEBSF 104 μM, aprotinin 80 nM, leupeptin 2 μM, bestatin 4 μM, pepstatin A 1.5 μM, and E-64 1.4 μM (0.1% of protease inhibitor cocktail P8340, Sigma-Aldrich, St. Louis, Mo.) at 4° C. The resuspended cells were homogenized using a Polytron (Brinkman, Westbury, N.Y.) at setting 6 for 10 sec., then centrifuged at 48,000×g for 10 min. The resulting pellet was washed by repeating the resuspension, homogenization and centrifugation steps. The final pellet was resuspended in buffer at 4° C. to yield a total protein concentration of 5 mg/ml, then aliquots were frozen in liquid nitrogen for further storage at −70° C. Immediately before carrying out a binding assay, an aliquot of cell homogenate was thawed and diluted to a concentration of 100 μg/ml in assay buffer consisting of 50 mM HEPES pH 5.0 and 0.1% CHAPSO. Assays were initiated in polypropylene 96-well plates (Costar, Cambridge, Mass.) by the addition of 200 μl of cell homogenate to 50 μl of assay buffer containing 1 nM radioligand (WO 2004 013098, compound 3, where the methoxy group is substituted for C(³H)₃: 80 Ci/mMol) and various concentrations of unlabelled compounds, and incubated for 1.5 hr. at 25° C. Separation of bound from free radioligand was by filtration on GFF glass fiber filters (Innotech Biosystems International, Lansing, Mich.) using an Innotech cell harvester. Filters were washed three times with 0.3 ml of phosphate buffered saline pH 7.0 at 4° C. and assessed for radioactivity using a Wallac 1450 Microbeta liquid scintillation counter (PerkinElmer, Boston, Mass.). Ki values of competing compounds were derived through Cheng-Prussoff correction of IC50 values calculated using XLfit (IDBS, Guildford, UK).

Abbreviations:

AEBSF; 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride

CHAPSO: 3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate

D-MEM: Dulbecco's modified eagle medium

TABLE 1 The activity of specific compounds described herein and tested in the above assay is provided in Table 1. Compounds Activity of Example Rating 1 ++ 2 ++ 3 + 4 + 5 + 6 + 7 + 8 + 9 ++ 10 ++ 11 ++ 12 ++ 13 + 14 ++ 15 ++ 16 ++ 17 + 18 + 19 + 20 +++ 21 ++ 22 ++ 23 +++ 24 +++ 25 +++ ^(a)Activity based on IC₅₀ values: +++ = <0.1 μM ++ = 0.1–1.0 μM + = >1.0 μM HEPES: 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid RAGE ™: Random Activation of Gene Expression ™ In Vitro Assay to Identify β-Secretase Inhibitor Based on the Inhibition of Aβ Formation from Membrane Preparations.

An isolated membrane fraction which contains functionally active β-secretase and β-APP substrates can generate β-secretase cleavage products including Aβ (Roberts, S. B.; Hendrick, J. P.; Vinitsky, A.; Lewis, M.; Smith, D. W.; Pak, R. PCT Publication WO 01/0175435; Fechteler, K.; Kostka, M.; Fuchs, M. Patent Application No. DE 99-19941039; Shearman, M.; Beher, D. et al., Biochemistry, 2000, 39, 8698-8704; Zhang, L. Song, L. et al., Biochemistry 2001, 40, 5049-5055). An isolated membrane fraction can be prepared from human derived cell lines such as HeLa and H4 which have been transfected with wild type or mutant forms of β-APP or a human alkaline phosphatase β-APP fusion construct, and stably express high levels of β-secretase substrates. The endogenous β-secretase present in the isolated membranes prepared at 0-4° C. cleaves the β-APP substrates when the membranes are shifted from 0-4 to 37° C. Detection of the cleavage products including Aβ can be monitored by standard techniques such as immunoprecipitation (Citron, M.; Diehl, T. S. et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 13170-13175), western blot (Klafki, H.-W.; Ambramowski, D. et al., J. Biol. Chem. 1996, 271, 28655-28659), enzyme linked immunosorbent assay (ELISA) as demonstrated by Seubert, P.; Vigo-Pelfrey, C. et al., Nature, 1992, 359, 325-327, or by a preferred method using time-resolved fluorescence of the homogeneous sample containing membranes and AD3 (Roberts, S. B.; Hendrick, J. P.; Vinitsky, A.; Lewis, M.; Smith, D. W.; Pak, R. PCT Publication WO 01/0175435; Shearman, M.; Beher, D. et al., Biochemistry, 2000, 39, 8698-8704). The Aβ present in a homogeneous sample containing membranes can be detected by time-resolved fluorescence with two antibodies that recognize different epitopes of Aβ. One of the antibodies recognizes an epitope that is present in Aβ but not present in the precursor fragments; preferably the antibody binds the carboxyl terminus of Aβ generated by the β-secretase cleavage. The second antibody binds to any other epitope present on Aβ. For example, antibodies that bind the N-terminal region (e.g., 26D6-B2-B3® SIBIA Neurosciences, La Jolla, Calif.) or bind the C-terminal end (e.g., 9S3.2® antibody, Biosolutions, Newark, Del.) of the Aβ peptide are known. The antibodies are labeled with a pair of fluorescent adducts that transfer fluorescent energy when the adducts are brought in close proximity as a result of binding to the N- and C-terminal ends or regions of Aβ. A lack of fluorescence is indicative of the absence of cleavage products, resulting from inhibition of β-secretase. The isolated membrane assay can be used to identify candidate agents that inhibit the activity of β-secretase cleavage and Aβ production.

A typical membrane-based assay requires 45 μg membrane protein per well in a 96- or 384-well format. Membranes in a neutral buffer are combined with the test compound and shifted from 0-4 to 37° C. Test agents may typically consist of synthetic compounds, secondary metabolites from bacterial or fungal fermentation extracts, or extracts from plant or marine samples. All synthetic agents are initially screened at doses ranging from 10-100 μM or in the case of extracts at sufficient dilution to minimize cytotoxicity. Incubation of the membranes with the test agent will continue for approximately 90 minutes at which time fluorescence labeled antibodies are added to each well for Aβ quantitation. The time-resolved fluorescence detection and quantitation of Aβ is described elsewhere (Roberts, S. B.; Hendrick, J. P.; Vinitsky, A.; Lewis, M.; Smith, D. W.; Pak, R. PCT Publication WO 01/0175435; Shearman, M.; Beher, D. et al., Biochemistry, 2000. 39, 8698-8704). Results are obtained by analysis of the plate in a fluorescence plate reader and comparison to the mock treated membranes and samples in which known amounts of Aβ were added to construct a standard concentration curve. A positive acting compound is one that inhibits the Aβ relative to the control sample by at least 50% at the initial tested concentration. Compounds of the present application are considered active when tested in the above assay if the IC₅₀ value for the test compound is less than 50 μM. A preferred IC₅₀ value is less than 1 μM. A more preferred IC₅₀ value is less than 0.1 μM. If a compound is found to be active then a dose response experiment is performed to determine the lowest dose of compound necessary to elicit the inhibition of the production of Aβ.

In Vivo Assays for the Determination of Aβ Reduction by a β-Secretase Inhibitor.

In vivo assays are available to demonstrate the inhibition of β-secretase activity. In these assays, animals, such as mice, that express normal levels of APP, β- and γ-secretase or are engineered to express higher levels of APP and hence Aβ can be used to demonstrate the utility of β-secretase inhibitors, as demonstrated with γ-secretase inhibitors [Dovey, H. et al., (2001), J. Neurochem. 76: 173-181]. In these assays, β-secretase inhibitors are administered to animals and Aβ levels in multiple compartments, such as plasma, cerebral spinal fluid, and brain extracts, are monitored for Aβ levels using methods previously outlined. For instance, Tg2576 mice, which overexpress human APP, are administered β-secretase inhibitors by oral gavage at doses that will cause measurable Aβ lowering, typically less than 100 mg/kg. Three hours after dosing plasma, brain, and CSF are collected, frozen in liquid nitrogen, and stored at −80° C. until analysis. For Aβ detection, plasma is diluted 15-fold in PBS with 0.1% Chaps while CSF is diluted 15-fold in 1% Chaps with protease inhibitors (5 μg/ml leupeptin, 30 μg/ml aprotirin, 1 mM phenylmethylsulfonylfluoride, 1 μM pepstatin). Brains are homogenized in 1% Chaps with protease inhibitors using 24 ml solution/g brain tissue. Homogenates were then centrifuged at 100,000×g for 1 hr at 4° C. The resulting supernatants were then diluted 10-fold in 1% Chaps with protease inhibitors. Aβ levels in the plasma, CSF, and brain lysate can then be measured using time-resolved fluorescence of the homogenous sample or one of the other methods previously described.

A β-secretase inhibitor is considered active in one of the above in vivo assays if it reduces Aβ by at least 50% at a dosage of 100 mg/kg.

Dosage and Formulation

The compounds of the present application can be administered orally using any pharmaceutically acceptable dosage form known in the art for such administration. The active ingredient can be supplied in solid dosage forms such as dry powders, granules, tablets or capsules, or in liquid dosage forms, such as syrups or aqueous suspensions. The active ingredient can be administered alone, but is generally administered with a pharmaceutical carrier. A valuable treatise with respect to pharmaceutical dosage forms is Remington's Pharmaceutical Sciences, Mack Publishing.

The compounds of the present application can be administered in such oral dosage forms as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. Likewise, they may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the compound desired can be employed to prevent or treat neurological disorders related to β-amyloid production or accumulation, such as Alzheimer's disease and Down's Syndrome.

The compounds of this application can be administered by any means that produces contact of the active agent with the agent's site of action in the body of a host, such as a human or a mammal. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents. They can be administered alone, but generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

The dosage regimen for the compounds of the present application will, of course, vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the condition.

Advantageously, compounds of the present application may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.

The compounds for the present application can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittant throughout the dosage regimen.

In the methods of the present application, the compounds herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients, or carriers (collectively referred to herein as carrier materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl callulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like; for oral administration in liquid form, the oral drug components can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or β-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

The compounds of the present disclosure can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamallar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.

Compounds of the present disclosure may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds of the present dislcosure may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.

Gelatin capsules may contain the active ingredient and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.

Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. 

1. A compound of Formula (I); or a stereoisomer thereof

wherein R₁ is phenyl optionally substituted with one or more groups selected from halogen, CN, CF₃, OH, —NH₂, —CH═CH-phenyl, pyridyl, thienyl, C₃₋₆cycloalkyl, C₁₋₆alkoxy, C₂₋₆alkenyl, OR₂₁, C₁₋₆alkyl optionally substituted with OH or —NH₂, —CH═CH—C₁₋₄alkyl optionally substituted with C₁₋₄alkoxy or OH, phenyl optionally substituted with halogen, —(CH₂)_(m)—NHC(═O)OC₁₋₆ alkyl optionally substituted with R₁₇, —(CH₂)_(m)—NHC(═O)Ophenyl optionally substituted with halogen, —(CH₂)_(m)—NHC(═O)R₁₈, —NH(C═O)R₁₉, —CH₂NH(SO₂)R₂₀, —CH₂morpholino and 4-methylpiperazinyl; R₂ and R₃ are each independently hydrogen, methyl or hydroxymethyl; m is 0 or 1; R₄ and R₅ are each independently hydrogen or methyl; R₆ is hydrogen, C₁₋₆alkyl, C₁₋₆alkyl NHC(═O) or CN; R₇ is hydrogen, C₁₋₆ alkyl, or phenylmethyl in which said phenyl is optionally substituted with one or more groups selected from halogen, CN, CF₃, and OCH₃; R₈ is hydrogen, halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, benzyloxy or CF₃; R₁₇ is C₁₋₄alkoxy, halogen, C₂₋₃alkynyl, 4-nitrophenyl, benzyloxy or benzothiophene 1,1-dioxide; R₁₈ is C₁₋₆alkyl or C₃₋₆cycloalkyl in which each are optionally substituted with a group selected from halogen, CN, CF₃, —N(CH₃)₂, C₁₋₄alkoxy, C₃₋₆cycloalkyl, morpholino, thienyl, imidazolyl, pyridyl, azepinyl, benzothienyl, benzofuranyl, phenyl and methoxyphenyl; R₁₉ is C₁₋₆alkyl, C₁₋₄alkoxy, C₃₋₆cycloalkyl optionally substituted with —N(CH₃)₂ or methylthio, phenyl, pyridyl optionally substituted with one or two halogen and C₁₋₄alkyl, benzothienyl, 1-methyl-2-pyrrolyl or furanyl; R₂₀ is C₁₋₆alkyl, 1,2-dimethyl-1H-imidazolyl, thienyl or phenyl in which said phenyl is optionally substituted with a group selected from acetyl, C₁₋₄alkoxy, benzyloxy, halogen, NH(C═O)CH₃ and nitro; and R₂₁ is C₁₋₆alkyl optionally substituted with a group selected from C₁₋₄alkoxy, halogen, phenyl, furanyl and methylamino; or a nontoxic pharmaceutically acceptable salt thereof.
 2. The compound according to claim 1 of Formula (1); or a stereoisomer thereof

wherein R₁ is phenyl optionally substituted with one or more groups selected from halogen, CN, CF₃, OH, —NH₂, —CH═CH-phenyl, pyridyl, thienyl, C₃₋₆cycloalkyl, C₁₋₆alkoxy, C₂₋₆alkenyl, OR₂₁, C₁₋₆alkyl optionally substituted with OH or —NH₂, —CH═CH—C₁₋₄alkyl optionally substituted with C₁₋₄alkoxy or OH, phenyl optionally substituted with halogen, —(CH₂)_(m)—NHC(═O)OC₁₋₆alkyl, —(CH₂)_(m)—NHC(═O)R₁₈, —NH(C═O)R₁₉, —CH₂NH(SO₂)R₂₀ and 4-methylpiperazinyl; R₂ and R₃ are hydrogen; m is 0 or 1; R₄ and R₅ are each independently hydrogen or methyl; R₆ is hydrogen, C₁₋₆alkyl, C₁₋₆alkyl NHC(═O) or CN; R₇ is hydrogen, C₁₋₆ alkyl, or phenylmethyl in which said phenyl is optionally substituted with one or more groups selected from halogen, CN, CF₃, or OCH₃; R₈ is hydrogen, halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, benzyloxy or CF₃; R₁₈ is C₁₋₆alkyl or C₃₋₆cycloalkyl in which each are optionally substituted with a group selected from halogen, CN, CF₃, —N(CH₃)₂, C₁₋₄alkoxy, C₃₋₆cycloalkyl, morpholino, thienyl, imidazolyl, pyridyl, azepinyl, benzothienyl, benzofuranyl, phenyl and methoxyphenyl; R₁₉ is C₁₋₆alkyl, C₁₋₄alkoxy, C₃₋₆cycloalkyl optionally substituted with —N(CH₃)₂ or methylthio, phenyl, pyridyl optionally substituted with one or two halogen and C₁₋₄alkyl, benzothienyl, 1-methyl-2-pyrrolyl or furanyl; R₂₀ is C₁₋₆alkyl, 1,2-dimethyl-1H-imidazolyl, thienyl or phenyl in which said phenyl is optionally substituted with a group selected from acetyl, C₁₋₄alkoxy, benzyloxy, halogen, NH(C═O)CH₃ and nitro; and R₂₁ is C₁₋₆alkyl optionally substituted with a halogen or phenyl; or a nontoxic pharmaceutically acceptable salt thereof.
 3. The compound according to claim 2 of Formula (1); or a stereoisomer thereof

wherein R₁ is phenyl optionally substituted with one or more groups selected from halogen, CN, CF₃, OH, —NH₂, C₃₋₆cycloalkyl, C₁₋₆alkoxy, C₂₋₆alkenyl, OR₂₁, C₁₋₆alkyl optionally substituted with OH or —NH₂, —CH═CH—C₁₋₄alkyl optionally substituted with C₁₋₄alkoxy or OH, —(CH₂)_(m)—NHC(═O)OC₁₋₆alkyl, —(CH₂)_(m)—NHC(═O)R₁₈, —NH(C═O)R₁₉, —CH₂NH(SO₂)R₂₀ and 4-methylpiperazinyl; R₂ and R₃ are hydrogen; m is 0 or 1; R₄ and R₅ are each independently hydrogen or methyl; R₆ is hydrogen, C₁₋₆alkyl, C₁₋₆alkyl NHC(═O) or CN; R₇ is hydrogen, C₁₋₆ alkyl, or phenylmethyl in which said phenyl is optionally substituted with one or more groups selected from halogen, CN, CF₃, or OCH₃; R₈ is hydrogen, halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, benzyloxy or CF₃; R₁₈ is C₁₋₆alkyl optionally substituted with a group selected from halogen, CN, CF₃, —N(CH₃)₂ and C₁₋₄alkoxy; R₁₉ is C₁₋₆alkyl, C₁₋₄alkoxy, C₃₋₆cycloalkyl optionally substituted with —N(CH₃)₂ or methylthio, phenyl or pyridyl optionally substituted with one or two halogen and C₁₋₄alkyl; R₂₀ is C₁₋₆alkyl, 1,2-dimethyl-1H-imidazolyl, thienyl or phenyl in which said phenyl is optionally substituted with a group selected from acetyl, C₁₋₄alkoxy, benzyloxy, halogen, NH(C═O)CH₃ and nitro; and R₂₁ is C₁₋₆alkyl optionally substituted with halogen or phenyl; or a nontoxic pharmaceutically acceptable salt thereof.
 4. The compound according to claim 3 of Formula (1); or a stereoisomer thereof

wherein R₁ is phenyl optionally substituted with one or more groups selected from halogen, CN, CF₃, OH, —NH₂, OR₂₁, C₁₋₆alkyl, NH(C═O)R₁₉ and 4-methylpiperazinyl; R₂ and R₃ are hydrogen; R₄ and R₅ are each independently hydrogen or methyl; R₆ is hydrogen, C₁₋₆alkyl, C₁₋₆alkyl NHC(═O) or CN; R₇ is hydrogen, C₁₋₆ alkyl, or phenylmethyl in which said phenyl is optionally substituted with one or more groups selected from halogen, CN, CF₃, or OCH₃; R₈ is hydrogen, halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, benzyloxy or CF₃; R₁₈ is C₁₋₆alkyl optionally substituted with —N(CH₃)₂; R₁₉ is C₁₋₆alkyl, C₁₋₄alkoxy, C₃₋₆cycloalkyl optionally substituted with —N(CH₃)₂ or methylthio, phenyl or pyridyl optionally substituted with one or two halogen and C₁₋₄alkyl; and R₂₁ is C₁₋₆alkyl optionally substituted with halogen or phenyl; or a nontoxic pharmaceutically acceptable salt thereof.
 5. The compound according to claim 1 selected from the group consisting of: N-(4-{N′-[2-(6-Bromo-1H-indol-3-yl)-acetyl]-guanidinomethyl}-2,6-dichloro-phenyl)-acetamide; N-(2,6-Dichloro-4-{N′-[2-(5-methoxy-1H-indol-3-yl)-acetyl]-guanidinomethyl}-phenyl)-acetamide; N-(3,5-Dichloro-benzyl)-N′-[2-(5-methoxy-1H-indol-3-yl)-acetyl]-guanidine; N-(3,5-Dichloro-benzyl)-N′-[2-(5-fluoro-1H-indol-3-yl)-acetyl]-guanidine; N-((4-amino-3,5-dichlorobenzylamino)(amino)methylene)-2-(1-methyl-1H-indol-3-yl)acetamide; N-((4-amino-3,5-dichlorobenzylamino)(amino)methylene)-2-(1-(4-fluorobenzyl)-1H-indol-3-yl)acetamide; N-((4-amino-3,5-dichlorobenzylamino)(amino)methylene)-2-(5-methoxy-1H-indol-3-yl)acetamide; N-((4-amino-3,5-dichlorobenzylamino)(amino)methylene)-2-(1H-indol-3-yl)propanamide; N-(2-Chloro-4-{N′-[2-(5-methoxy-1H-indol-3-yl)-acetyl]-guanidinomethyl}-6-methyl-phenyl)-acetamide; N-(2,6-Dichloro-4-{N′-[2-(5-methoxy-2-methyl-1H-indol-3-yl)-acetyl]-guanidinomethyl}-phenyl)-acetamide; N-(2,6-Dichloro-4-{N′-[2-(2-methyl-1H-indol-3-yl)-acetyl]-guanidinomethyl}-phenyl)-acetamide; N-(2,6-Dichloro-4-{N′-[2-(5-fluoro-1H-indol-3-yl)-acetyl]-guanidinomethyl}-phenyl)-acetamide; N-(4-{N′-[2-(5-Benzyloxy-1H-indol-3-yl)-acetyl]-guanidinomethyl}-2,6-dichloro-phenyl)-acetamide; N-(2,6-Dichloro-4-{N′-[2-(5-hydroxy-1H-indol-3-yl)-acetyl]-guanidinomethyl}-phenyl)-acetamide; N-(4-{N′-[2-(6-Benzyloxy-1H-indol-3-yl)-acetyl]-guanidinomethyl}-2,6-dichloro-phenyl)-acetamide; N-(2,6-Dichloro-4-{N′-[2-(6-fluoro-1H-indol-3-yl)-acetyl]-guanidinomethyl}-phenyl)-acetamide; N-(4-Amino-3,5-dichloro-benzyl)-N′-[2-(5-bromo-1H-indol-3-yl)-acetyl]-guanidine; N-[2-(5-Bromo-1H-indol-3-yl)-acetyl]-N′-[4-(4-methyl-piperazin-1-yl)-benzyl]-guanidine; N-[2-(5-Bromo-1H-indol-3-yl)-acetyl]-N′-[3-chloro-4-(4-methyl-piperazin-1-yl)-benzyl]-guanidine; N-(4-{N′-[2-(5-Bromo-1H-indol-3-yl)-acetyl]-guanidinomethyl}-2,6-dichloro -phenyl)-acetamide; N-(2,6-Dichloro-4-{N′-[2-(5-fluoro-2-methyl-1H-indol-3-yl)-acetyl]-guanidinomethyl}-phenyl)-acetamide; N-(2,6-Dichloro-4-{N′-[2-(7-fluoro-2-methyl-1H-indol-3-yl)-acetyl]-guanidinomethyl}-phenyl)-acetamide; N-(4-{N′-[2-(5-Cyano-1H-indol-3-yl)-acetyl]-guanidinomethyl}-2,6-dichloro -phenyl)-acetamide; 3-{2-[N′-(4-Acetylamino-3,5-dichloro-benzyl)-guanidino]-2-oxo-ethyl}-5-bromo-1H-indole-2-carboxylic acid tert-butylamide; and N-(4-{N′-[2-(5-Bromo-2-cyano-1H-indol-3-yl)-acetyl]-guanidinomethyl}-2,6-dichloro-phenyl)-acetamide; or a nontoxic pharmaceutically acceptable salt thereof.
 6. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1 in association with a pharmaceutically acceptable adjuvant, carrier or diluent. 